Alphaherpesviruses are major human and animal pathogens that cause a wide spectrum of disease in the infected host. Us3 is a serine/threonine kinase, encoded by all alphaherpesviruses, which functions in the prevention of virus-induced apoptosis as well as de-envelopment of primary enveloped virions from the perinuclear space during virion maturation. In addition, Us3 plays an important role in the cell-to-cell spread of virus infection, which has been a focus of our studies. Cell-to-cell spread of infection is a critically important mechanism for virus dissemination in the host for many viruses and this is particularly true for the herpesviruses. Reactivation of herpesviruses occurs in the presence of a fully primed immune system. Cell-to-cell spread of virus infection occurs quickly and can continue in the presence of neutralizing antibodies. Re-organization of the actin cytoskeleton through Us3 kinase activity is required for Us3 enhancement of cell-to-cell spread. The identification of cellular substrates for Us3 that facilitate virus spread would represent a major advance in our understanding of this process and has the potential to identify cellular targets for antiviral interventions. Using a yeast two-hybrid substrate trap we identified the cellular protein, Eps8 (epidermal growth factor receptor protein substrate 8), as a potential Us3 substrate. Eps8 is a signaling molecule required for actin remodeling in response to growth factor stimulation. Our preliminary data indicate that Us3 and Eps8 interact in animal cells, that Eps8 is required for Us3-mediated actin remodeling and that both Eps8 and Us3 are required for efficient spread of infection between cells. In this application, we propose to define the molecular basis for the interaction between Us3 and Eps8, determine how this interaction promotes cell-to-cell spread of virus infection and examine the consequences of this interaction in vivo. Aim 1: define the molecular basis for the interaction between Us3 and Eps8. Aim 2: define the mechanism by which Us3 directs Eps8 activities. Aim 3: determine the significance of the Us3/Eps8 interaction to virus pathogenesis.